CN101464463A - Front S1 and front S2 combined detection reagent kit for hepatitis B virus and method for producing the same - Google Patents
Front S1 and front S2 combined detection reagent kit for hepatitis B virus and method for producing the same Download PDFInfo
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- CN101464463A CN101464463A CNA2007101725713A CN200710172571A CN101464463A CN 101464463 A CN101464463 A CN 101464463A CN A2007101725713 A CNA2007101725713 A CN A2007101725713A CN 200710172571 A CN200710172571 A CN 200710172571A CN 101464463 A CN101464463 A CN 101464463A
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- liquid
- antibody
- colour developing
- hepatitis
- substrate colour
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Abstract
The invention provides a reagent kit for combined detection of pre-S1 and pre-S2 of hepatitis B virus, which comprises a coating reaction strip (with 48 holes or 96 holes), an enzyme labeled antibody, a positive control liquid, a negative control liquid, a concentrated scrub solution, a substrate chromogenic A liquid, a substrate chromogenic B liquid and a stopping solution. The reagent kit can detect a pre-S1 antigen and a pre-S2 antigen at the same time, the use is convenient, and the result is accurate.
Description
Technical field:
The present invention relates to the biologic product technology field.Be specifically related to hepatitis B virus front S 1 (preS1) antigen and preceding S2 (preS2) antigen enzyme linked immunological combined detection kit and preparation method thereof.
Background technology:
Liver is one of vitals that earn a bare living, and one of greatest factor of liver damage is a virus infections.So far, identified the hepatitis virus of five kinds of dissimilar impaired livers such as first type, B-mode, third type, fourth type and hepatitis E virus.Wherein, liver cell develops into chronic hepatitis and lasting virus replication can cause the possibility of cirrhosis always by people's broad research owing to hepatitis B (HBV) destroys.
Hepatitis type B virus (HBV) is the part double-spiral structure of a ring-type, is about 3.2kb.HBV genome encoding HBV antigen, all four functional single open reading frames are positioned at the DNA minus strand, P gene code archaeal dna polymerase.C gene code HbcAg.S gene code S antigen, pre S 1 antigen and preS 2 antigen.X gene coding X product.Belong to same frame at two little ORFs of S gene front (open reading frame open reading frame) and S gene ORF, ORFS can be readed over down, the antigen that the two kinds of S albumen of encoding are relevant, these two kinds of antigens also are present in the surface of virion, before these two antigens are called-and S1 (pre-S1) and preceding-S2 (pre-S2).Equally, a short ORF is arranged also in the ORFC front, before being called-C (pre-C), the bigger C albumen related antigen of encoding.All on minus-strand dna (long-chain), wherein the S gene is overlapped in the pol gene all these ORF fully, X gene and pol gene, C gene overlap, and C gene and polymerase also have overlapping.
Hepatitis b virus s antigen (HBsAg) is one group of albumen that constitutes outer virionic membrane, and one of its critical function is the acceptor on the identification liver plasma membrane, makes virus and cells contacting, invades in the born of the same parents then and duplicates.HBsAg contains big or middle, and little 3 kinds of molecular forms protein, they are all coded by the S-open reading frame (S-ORF) of viral gene.Wherein the promptly main albumen of small molecular protein (SHBs) is made up of 226 amino acid, middle albumen (MHBs) is to add 55 amino acid whose preS2 districts at main albumen n end, the preS1 district that is made up of 108 or 119 amino acid was not added in the N end front of albumen during high molecular weight protein (LHBs) did not coexist according to the hypotype of virus, and these 3 kinds of protein moleculars all exist glycosylation and nonglycosylated form.Separate the virion surface obtain different shape from serum, SHBs content is the highest, but MHBs and LHBs content are different, and in the spherical ghost particle of 22nm, LHBs content is extremely low, and the content that contains LHBs in the complete virion of DNA can reach 20%.
HBV-DNA detection is used as the goldstandard that HBV infects and duplicates usually, and available PCR method is measured HBV-DNA, but that the PCR method operation requires is high, cost is high, need higher experiment condition, still can not carry out and makes its application limited at many different medical units.Tradition is thought in hepatitis B in detecting serum " two double " index, HBeAg turns out cloudy and represents stopping of hbv replication, but in the chronic hepatitis B patient of HBeAg feminine gender, when if the terminator codon sudden change takes place in preceding c district, maybe when being positioned at a c promoter that x reads sign indicating number district and undergoing mutation, HBeAg expresses and will stop or reduce, cause the HBeAg dyssynthesis, HBeAg is negative, but HBV DNA is still positive, virus replication is not affected, and illustrates that the HBeAg feminine gender can not stop desirable basis for estimation as hbv replication, and the immunoserology index of therefore exploring new judgement hbv replication has become one of present research focus.
The clinical meaning and the purposes of pre S 1 antigen (Pre-S1Ag): 1, the infection of reflection HBV and the index of duplicating situation, a lot of bibliographical information (Liu Tuo etc., hepatitis B virus front S 1 Protein Detection and clinical meaning, the Ningxia medical journal, 2006,28:204-205) (happy love equality, HBVpreS1-Ag, HBV-DNA, the correlation analysis of HBVM and meaning thereof, Jiangxi Medical College's journal, 2004,44:27-30) pre S 1 antigen can be used as replenishing and contrast that HbeAg and HBV-DNA detect, can remedy " mistake is sought " of the HBeAg (-) that due to illness poison variation and other reason cause.2, be applied to the judgement of prognosis and curative effect of medication, acute hepatitis B patient pre S 1 antigen is cloudy to be changeed more early, prognosis is good more, be the sign the earliest of virus sweep, otherwise, the pre S 1 antigen lasting masculin, to be developed to chronic hepatitis, due to illness HBeAb (+) chronic hepatitis B patients of virus gene variation, easily progress is cirrhosis, even liver cancer.Strengthening detecting pre S 1 antigen, is a kind of excellent means that detects disease prognosis.3, the infection of early diagnosis hepatitis type B virus, pre S 1 antigen appear at the very early time that acute hepatitis B infects, and can find before transaminase raises, and prompting can be used as the early diagnosis hepatitis B virus infection.In health check-up and blood donor, add and look into pre S 1 antigen, the early diagnosis of the disease of can getting up and cut off the vital role of infectious agent as early as possible.
The binding site that the polyerized human serum albumin is arranged on the preS 2 antigen (Pre-S2) participates in the process of HBV infected liver cell, adheres to and invade in the hepatocellular mechanism at HBV to play an important role.Bibliographical information also can have a FFI special and responsive observation index as the asymptomatic carrier of HbsAg with Pre-S2.(Yu Shu is high, and HbsAg asymptomatic carrier's serum HBV DNA and PreS2 concern pre-test, Fujian Medical College's journal, and 1992,26:290-292).
S1, preS 2 antigen are not only the new sign that HBV infects before the hepatitis B, also can be used as the index of the prognosis judgement of virus replication and acute and chronic hepatitis B, the continuous monitoring of HBV PreS1-Ag, PreS2-Ag also is the good observation index of immune clearance HBV, antiviral therapy and disease prognosis.
Summary of the invention:
The technical problem to be solved in the present invention is to overcome above-mentioned weak point, a kind of kit that can detect pre S 1 antigen and preS 2 antigen simultaneously of research and design.
The invention provides a kind of hepatitis B virus front S 1 and preceding S2 combined detection kit.
Each constituent comprises in the kit: antibody sandwich reaction bar 48 or 96 holes, enzymic-labelled antibody working fluid 1 bottle of (3.5ml or 7ml), positive control solution 1 (0.5ml or 1ml), 1 (0.5ml or 1ml) of negative controls, 20 times of concentrated cleaning solutions 1 bottle of (10ml or 24ml), substrate colour developing A liquid 1 bottle of (3.5ml or 7ml), substrate colour developing B liquid 1 bottle of (3.5ml or 7ml) and 1 bottle of stop buffer (3.5ml or 7ml).
Positive control preparation: the serum of the hepatitis B patient that HbeAg and HBV-DNA are positive simultaneously.
The preparation of negative control: normal human serum.
Substrate colour developing A liquid: Na
2HPO
412H
2O (1.7g/100ml), citric acid (0.5g/100ml), 3%H
2O
2(200ul/100ml).
Substrate colour developing B liquid: Na
2HPO
412H
2O (1.7g/100ml), citric acid (0.5g/100ml), tetramethyl benzidine (0.15g/100ml).
Stop buffer: 2mol/L H
2SO
4
20 times of concentrated cleaning solution: Na
2HPO
412H
2O (2.6g/100ml), NaH
2PO
42H
2O (0.4g/100ml), 20% Tween-20 (2.5ml/100ml).
Enzymic-labelled antibody working fluid: Na
2HPO
412H
2O (1.86g/L), KH
2PO
42H
2O (0.22g/L), NaCl (9g/L), bovine serum albumin(BSA) (10g/L) reach by suitable concn and dilute good enzymic-labelled antibody.
Antibody sandwich reaction bar: coated antibody joins the carbonate buffer solution (Na for preparing with 1-10ug/ml
2CO
3: 1.6g/L, Na
2HCO
3: 2.93g/L), join in the microwell plate with the 100ul/ hole then, put standing over night drying in 4 ℃ of wet boxes.
Another object of the present invention has provided the preparation method of above-mentioned hepatitis B virus front S 1 and preceding S2 combined detection kit.
(1): adopt the HbsAg of genetic recombination to prepare HbsAb coated antibody or enzymic-labelled antibody;
(2): adopt the preS1Ag of genetic recombination, the anti-preceding S1 of preceding S2 polypeptide preparation, preceding S2 coated antibody or enzymic-labelled antibody;
(3): antiserum or ascites obtain pure antibody with ammonium sulfate precipitation, affinity column (ProteinA/G) or ion exchange chromatography (DEAE-Sepharose) purifying;
(4): the pre-bag of preparation is by plate: antibody do bag by, add coating buffer, add reaction plate, 4 ℃ of wet boxes place spend the night, pat dry, seal, dry, pack;
(5): preparation enzyme labeling thing: enzymic-labelled antibody adds in the enzyme mark dilution with suitable concentration.
(6): with all the other compositions in the kit: negative control, positive control, substrate colour developing A liquid, substrate colour developing B liquid, stop buffer and concentrated cleaning solution are divided in the bottle.
Described substrate colour developing A liquid is: Na
2HPO
412H
2O, citric acid, 3%H
2O
2With redistilled water pH5.0.
Described substrate colour developing B liquid is: Na
2HPO
412H
2O, citric acid, 3% H
2O
2, TMB (tetramethyl benzidine) and redistilled water pH5.0.
Described stop buffer is: dense H
2SO
4And redistilled water.
Described 10 times of concentrated cleaning solution: Na
2HPO
412H
2O, NaH
2PO
42H
2O, 20% Tween-20 and redistilled water.
Effect of the present invention:
1, joint-detection (comprising that merging detects and separate detection, and detect simultaneously and successively detect that preferred while, merging detect) hepatitis B virus pre S 1 antigen and preS 2 antigen in sample with any order.
2, in same reaction container (as reacting hole), in identical reaction, detect hepatitis B virus front S 1 and preS 2 antigen simultaneously.
3, sample is the biological sample from the separation of individuality to be checked, as serum etc.
4, coated antibody is anti-HbsAg monoclonal antibody, and then the enzyme labeling bond is anti-preceding S1, preceding S2 mixed antibody, if coated antibody is anti-preceding S1, preceding S2 mixed antibody, then the enzyme labeling bond is anti-HbsAg monoclonal antibody.
Embodiment:
S1 and preceding S2 enzyme are exempted from the production stage of kit before the embodiment 1 preparation joint investigating hepatitis B virus
(1): Antibody Preparation
1. prepare the HBsAb monoclonal antibody: the HbsAg of genetic recombination adds freund adjuvant immunity Balb/C mouse, get positive mice spleen cell and myeloma cell and be fused into hybridoma, the screening positive clone cell line is beaten ascites and is got antibody, uses the affinity column purifying behind the first ammonium sulfate precipitation.
2. preparation former S 1 immune body: the preS1 Ag (1-119aa) of genetic recombination adds the freund adjuvant immune rabbit, adds isopyknic saturated (NH
4)
2SO
4Precipitation is used the ion-exchange chromatography purifying after slightly carrying again.
3. preparation pre-s2 antibody: S2 polypeptide (1-55aa) adds the freund adjuvant immune rabbit before synthetic, adds isopyknic saturated (NH
4)
2SO
4Precipitation is used the ion-exchange chromatography purifying after slightly carrying again.
(2): preparation enzyme labeling thing
The HBAb monoclonal antibody of purifying or preceding S1, preceding S2 mixed antibody are with simple and easy sodium periodate method and horseradish peroxidase (HRP) coupling.
(1) taking by weighing 5mgHRP is dissolved in the 1ml distilled water.
(2) in last liquid, add the 0.1M NaIO that 0.2ml newly joins
4Solution, lucifuge stirred 20 minutes under the room temperature.
(3) above-mentioned solution is packed in the bag filter, to the sodium-acetate buffer dialysis of 1mM PH4.4,4 ℃ are spent the night.
(4) add 20ul 0.2M PH9.5 carbonate buffer solution, make the PH of above hydroformylation HRP be elevated to 9.0～9.5, add 10mg IgG then immediately in 1ml 0.01M carbonate buffer solution, the room temperature lucifuge stirred 2 hours gently.
(5) add the 4mg/ml NaBH that 0.1ml newly joins
4Liquid, mixing, put again 4 ℃ 2 hours.
(6) above-mentioned liquid is packed in the bag filter, to 0.15M PH7.4 PBS dialysis, 4 ℃ are spent the night.
(7) under agitation dropwise add the equal-volume saturated ammonium sulfate, put 4 ℃ 1 hour.
(8) 3000rpm centrifugal half an hour, abandon supernatant.Sediment is washed secondary with semi-saturation ammonium sulfate, and last sediment is dissolved among the PBS of a small amount of 0.15M PH7.4.
(9) above-mentioned solution is packed in the bag filter, to the PB buffer saline dialysis of 0.15M PH7.4, after the dialysis fully, 10,000rpm removed precipitation in centrifugal 30 minutes, and supernatant is enzyme conjugates, and after the packing ,-20 ℃ of packing are preserved.
(3): the antibody preference pattern
Coated antibody is anti-HbsAg monoclonal antibody, and then the enzyme labeling bond is anti-preceding S1, preceding S2 mixed antibody, if coated antibody is anti-preceding S1, preceding S2 mixed antibody, then the enzyme labeling bond is anti-HbsAg monoclonal antibody.
The preparation of embodiment 2 kits
(1): the preparation of pre-coated antibody plate
1. bag quilt
Coated antibody joins in the carbonate buffer solution (pH:9.0-9.5) for preparing with 1-10ug/ml, joins in the microwell plate with the 100ul/ hole then, puts standing over night drying in 4 ℃ of wet boxes.
2. sealing
In coated slab, add the confining liquid in 200ul/ hole, 37 ℃ of sealings 2 hours dry, to be dried after, put into the polybag of drying agent, seal, in 4 ℃, preserve.
3. enzyme labeling thing: enzymic-labelled antibody adds in the enzyme mark dilution with suitable concentration (1:1000).
4. positive control preparation: the serum of the hepatitis B patient that HbeAg and HBV-DNA are positive simultaneously, placed 1 hour for 60 ℃, aseptic filtration is with this kit measurement OD value〉0.5, packing.
5. the preparation of negative control: with this kit measurement normal human serum OD value between 0-0.05, packing.
6. substrate colour developing A liquid:
Na
2HPO
4·12H
2O 1.7g
Citric acid 0.5g
3％?H
2O
2 200
Redistilled water 100ml
Adjust pH:5.0
7. substrate colour developing B liquid:
Na
2HPO
4·12H
2O 1.7g
Citric acid 0.5g
Redistilled water 100ml
After adjusting pH to 5.0, add the solution 25ul that 10ml DMO contains 60mg TMB (tetramethyl benzidine).
8. stop buffer:
Dense H
2SO
411ml
Redistilled water 89ml
9.10 concentrated cleaning solution doubly:
Na
2HPO
4·12H
2O 2.6g
NaH
2PO
4·2H
2O 0.4g
20％?Tween-20 2.5ml
Redistilled water 100ml
Adjust pH:7.20
Embodiment 3 kits use
1, get the lath of aequum, every hole adds the 100ul sample.Each plate is established positive control 2 holes (100ul), negative control 2 holes (100ul), and 1 hole, blank hole (distilled water 100ul), 37 ℃ of incubations 30 minutes;
2, wash plate: dry liquid in the plate, add 400ul/ hole cleansing solution, place drying in 30 seconds, so repeat to wash five times;
3, except that the blank hole, each hole adds the enzymic-labelled antibody in 100ul/ hole, 37 ℃ of incubations 30 minutes;
4, wash plate: dry liquid in the plate, add 400ul/ hole cleansing solution, place drying in 30 seconds, so repeat to wash five times;
5, add substrate, 50ul/ hole TMB colour developing A liquid, 50ul/ hole TMB colour developing B liquid, mixing, 37 ℃ of incubations 15 minutes;
6, add 50ul/ hole stop buffer, put in the microplate reader under the 450nm wavelength, measure reading.
Claims (4)
1, a kind of hepatitis B virus front S 1 and preceding S2 combined detection kit is characterized in that it comprises antibody sandwich reaction bar, enzymic-labelled antibody, positive control solution, negative controls, concentrated cleaning solution, substrate colour developing A liquid, substrate colour developing B liquid and stop buffer.
2, antibody sandwich reaction bar according to claim 1 is 48 or 96 holes; 1 bottle of 3.5ml of enzymic-labelled antibody working fluid or 7ml; 1 0.5ml of positive control solution or 1ml, 1 0.5ml of negative controls or 1ml, 1 bottle of 10ml of 20 times of concentrated cleaning solutions or 24ml, substrate colour developing 1 bottle of 3.5ml of A liquid or 7ml, substrate colour developing 1 bottle of 3.5ml of B liquid or 7ml and 1 bottle of 3.5ml of stop buffer or 7ml.
3, the preparation method of a kind of hepatitis B virus front S 1 and preceding S2 combined detection kit is characterized in that this method comprises the following steps:
(1): adopt the HbsAg of genetic recombination to prepare HbsAb coated antibody or enzymic-labelled antibody;
(2): adopt the preS 1Ag of genetic recombination, the anti-preceding S1 of preceding S2 polypeptide preparation, preceding S2 coated antibody or enzymic-labelled antibody;
(3): antiserum or ascites obtain pure antibody with ammonium sulfate precipitation, affinity column or ion exchange chromatography purifying;
(4): the pre-bag of preparation is by plate: antibody do bag by, add coating buffer, add reaction plate, 4 ℃ of wet boxes place spend the night, pat dry, seal, dry, pack;
(5): preparation enzyme labeling thing: enzymic-labelled antibody adds in the enzyme mark dilution with suitable concentration;
(6): with all the other compositions in the kit: negative control, positive control, substrate colour developing A liquid, substrate colour developing B liquid, stop buffer and concentrated cleaning solution are divided in the bottle.
4, the preparation method of a kind of hepatitis B virus front S 1 according to claim 3 and preceding S2 combined detection kit is characterized in that the described substrate colour developing of step (6) A liquid is: Na
2HPO
412H
2O, citric acid, 3%H
2O
2With redistilled water pH5.0; Described substrate colour developing B liquid is: Na
2HPO
412H
2O citric acid, 3%H
2O
2, TMB (tetramethyl benzidine) and redistilled water pH5.0; Described stop buffer is: dense H
2SO
4And redistilled water; Described 10 times of concentrated cleaning solutions are: Na
2HPO
412H
2O, 20%Tween-20 and redistilled water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101725713A CN101464463A (en) | 2007-12-19 | 2007-12-19 | Front S1 and front S2 combined detection reagent kit for hepatitis B virus and method for producing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101725713A CN101464463A (en) | 2007-12-19 | 2007-12-19 | Front S1 and front S2 combined detection reagent kit for hepatitis B virus and method for producing the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101464463A true CN101464463A (en) | 2009-06-24 |
Family
ID=40805140
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007101725713A Pending CN101464463A (en) | 2007-12-19 | 2007-12-19 | Front S1 and front S2 combined detection reagent kit for hepatitis B virus and method for producing the same |
Country Status (1)
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|---|---|
| CN (1) | CN101464463A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108271358A (en) * | 2014-12-10 | 2018-07-10 | 财团法人卫生研究院 | Judge the antibody lacked in HBV virus pre-S2 regions and its method |
| CN108344869A (en) * | 2018-02-05 | 2018-07-31 | 苏州长光华医生物医学工程有限公司 | A kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit and its application |
| CN117214434A (en) * | 2023-08-14 | 2023-12-12 | 吉林大学 | HBV PreS1+PreS2 region protein-based typing kit and preparation method and application thereof |
| CN117214434B (en) * | 2023-08-14 | 2024-05-14 | 吉林大学 | HBV PreS1+PreS2 region protein-based typing kit and preparation method and application thereof |
-
2007
- 2007-12-19 CN CNA2007101725713A patent/CN101464463A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108271358A (en) * | 2014-12-10 | 2018-07-10 | 财团法人卫生研究院 | Judge the antibody lacked in HBV virus pre-S2 regions and its method |
| CN108344869A (en) * | 2018-02-05 | 2018-07-31 | 苏州长光华医生物医学工程有限公司 | A kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit and its application |
| CN117214434A (en) * | 2023-08-14 | 2023-12-12 | 吉林大学 | HBV PreS1+PreS2 region protein-based typing kit and preparation method and application thereof |
| CN117214434B (en) * | 2023-08-14 | 2024-05-14 | 吉林大学 | HBV PreS1+PreS2 region protein-based typing kit and preparation method and application thereof |
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Application publication date: 20090624 |